Bottom Line:
Yet the purpose of this remarkable structure has been an enigma.In addition, insect cells that naturally harbor dispersed Golgi stacks have limited capacity to transport artificial oversized cargoes.These results imply that the ribbon structure is an essential requirement for transport of large cargoes in mammalian cells, and we suggest that this is because it enables the dilated rims of cisternae (containing the aggregates) to move across the stack as they transfer among adjacent stacks within the ribbon structure.

Figure 1: Nocodazole disrupts the ribbon and inhibits aggregates secretion. (A) Confocal micrographs illustrate nocodazole-induced Golgi ribbon disruption. HeLa cells expressing GT-GFP were treated or not with nocodazole (2 h, 1 μg/ml) before being prepared for confocal imaging. (B) Schematic representation of the drug-controlled aggregate. The human growth hormone (hGH) is fused to a signal sequence (ss), a GFP, four repeats of the self-aggregating FM domain (FM4), and a furin cleavage site that is cleaved during flow through the trans-Golgi network (TGN). (C) Immunoblots show the inhibition of aggregate secretion under nocodazole treatment. Cells expressing ssGFP-FM4-hGH were pretreated with nocodazole (2 h), which was maintained during the rest of the experiment. Then the temperature was shifted to 16°C in the presence of the disaggregating drug (1 h and 30 min, 1 μM). When required, the drug was removed to trigger reaggregation (30 min). Media was removed and replaced with fresh media containing or not the disaggregating drug. Temperature was shifted to 37°C and the media were collected at the indicated time points and submitted for TCA precipitation while the cells were harvested. Unless mentioned otherwise, all chase experiments were performed in the presence of CHX. TCA-precipitated media and cell lysate fractions were analyzed by SDS–PAGE/immunoblotting, which was followed by densitometry measurement. (D) Graph represents the percent of secretion over time for each condition. Data represent the mean ± SD of three independent experiments.

Mentions:
First, we combined nocodazole treatment with a drug-controlled aggregation system that allows for positioning soluble aggregates at different stages of the secretory pathway (Volchuk et al., 2000; Lavieu et al., 2013). The chimeric cargo is constituted of a signal sequence fused to a fluorescent protein, four repeats of the self-aggregation domain (FM4), a furin cleavage site, and the sequence encoding the human growth hormone (Figure 1B). In the absence of the disaggregating drug, the chimeric protein is aggregated and stays in the endoplasmic reticulum (ER). In the presence of the drug, at 16°C for 2 h, the now-disaggregated protein reaches the cis face of the Golgi, where it can be reaggregated by subsequent drug removal. Shifting the temperature back to 37°C in the absence or presence of the disaggregating drug allows for comparison of intra-Golgi transport and further secretion of large aggregated cargo versus small disaggregated cargo, respectively. Importantly, such a chase needs to be performed in the presence of cycloheximide (CHX), an inhibitor of protein synthesis.

Figure 1: Nocodazole disrupts the ribbon and inhibits aggregates secretion. (A) Confocal micrographs illustrate nocodazole-induced Golgi ribbon disruption. HeLa cells expressing GT-GFP were treated or not with nocodazole (2 h, 1 μg/ml) before being prepared for confocal imaging. (B) Schematic representation of the drug-controlled aggregate. The human growth hormone (hGH) is fused to a signal sequence (ss), a GFP, four repeats of the self-aggregating FM domain (FM4), and a furin cleavage site that is cleaved during flow through the trans-Golgi network (TGN). (C) Immunoblots show the inhibition of aggregate secretion under nocodazole treatment. Cells expressing ssGFP-FM4-hGH were pretreated with nocodazole (2 h), which was maintained during the rest of the experiment. Then the temperature was shifted to 16°C in the presence of the disaggregating drug (1 h and 30 min, 1 μM). When required, the drug was removed to trigger reaggregation (30 min). Media was removed and replaced with fresh media containing or not the disaggregating drug. Temperature was shifted to 37°C and the media were collected at the indicated time points and submitted for TCA precipitation while the cells were harvested. Unless mentioned otherwise, all chase experiments were performed in the presence of CHX. TCA-precipitated media and cell lysate fractions were analyzed by SDS–PAGE/immunoblotting, which was followed by densitometry measurement. (D) Graph represents the percent of secretion over time for each condition. Data represent the mean ± SD of three independent experiments.

Mentions:
First, we combined nocodazole treatment with a drug-controlled aggregation system that allows for positioning soluble aggregates at different stages of the secretory pathway (Volchuk et al., 2000; Lavieu et al., 2013). The chimeric cargo is constituted of a signal sequence fused to a fluorescent protein, four repeats of the self-aggregation domain (FM4), a furin cleavage site, and the sequence encoding the human growth hormone (Figure 1B). In the absence of the disaggregating drug, the chimeric protein is aggregated and stays in the endoplasmic reticulum (ER). In the presence of the drug, at 16°C for 2 h, the now-disaggregated protein reaches the cis face of the Golgi, where it can be reaggregated by subsequent drug removal. Shifting the temperature back to 37°C in the absence or presence of the disaggregating drug allows for comparison of intra-Golgi transport and further secretion of large aggregated cargo versus small disaggregated cargo, respectively. Importantly, such a chase needs to be performed in the presence of cycloheximide (CHX), an inhibitor of protein synthesis.

Bottom Line:
Yet the purpose of this remarkable structure has been an enigma.In addition, insect cells that naturally harbor dispersed Golgi stacks have limited capacity to transport artificial oversized cargoes.These results imply that the ribbon structure is an essential requirement for transport of large cargoes in mammalian cells, and we suggest that this is because it enables the dilated rims of cisternae (containing the aggregates) to move across the stack as they transfer among adjacent stacks within the ribbon structure.